Preparation method and application of astragaloside-rADSCs hydrogel

Abstract

The invention provides a preparation method of astragaloside-rADSCs hydrogel, which comprises the following steps: extracting rADSCs, culturing the rADSCs to a P3 generation, co-culturing the rADSCs cultured to the P3 generation and an astragaloside solution, and collecting supernate to obtain the astragaloside-rADSCs; the preparation method comprises the following steps: uniformly mixing PF-127 with sodium hyaluronate, then mixing with astragaloside-rADSCs, stirring, and standing, so as to obtain the astragaloside-rADSCs hydrogel. The invention further provides an application of the prepared astragaloside-rADSCs hydrogel. The astragaloside-rADSCs hydrogel is used for preparing a medicine for repairing diabetic skin injury. According to the astragaloside-rADSCs hydrogel prepared by the invention, the astragaloside is used for regulating the stem cell function and enhancing the repair promotion capability of the stem cell, serum-related risks are avoided through serum-free culture, controlled release of cell factors is realized by virtue of the temperature-sensitive hydrogel, and a brand new non-invasive, non-drug-resistant and efficient repair treatment scheme is provided for diabetic skin injury.

Description

Technical Field

[0001] This invention belongs to the field of adipose-derived mesenchymal stem cell technology, specifically relating to a method for preparing and applying astragaloside A-rADSCs hydrogel. Background Technology

[0002] Current treatment of diabetic skin lesions faces numerous bottlenecks: in conventional treatments, blood glucose control drugs are prone to causing adverse reactions, anti-infective drugs have insufficient penetration into deep lesions, and drugs that improve microcirculation have limited effectiveness in patients with severe vascular disease, making it difficult to fundamentally repair the wound; simple adipose-derived mesenchymal stem cell (AD-MSCs) transplantation suffers from low in vivo survival rates and insufficient homing efficiency, affecting the repair effect. Furthermore, traditional AD-MSCs culture relies on culture media containing fetal bovine serum, which carries risks such as exogenous viral contamination, batch-to-batch inconsistencies in activity, and allergic reactions. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for preparing astragaloside A-rADSCs hydrogel and its application, which addresses the shortcomings of the prior art. The astragaloside A-rADSCs hydrogel prepared by this method utilizes astragaloside A to regulate stem cell function and enhance its repair ability. It also uses a thermosensitive hydrogel to achieve controlled release of cytokines, providing a new treatment solution for diabetic skin damage that is non-invasive, drug-resistant, and highly effective in repair.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing astragaloside A-rADSCs hydrogel, the method being as follows: S1. Rabbit adipose mesenchymal stem cells were extracted and cultured to generation P3 to obtain generation P3 rADSCs; S2. The P3 generation rADSCs obtained in S1 were co-cultured with astragaloside A solution, and the supernatant was collected to obtain astragaloside A-rADSCs. S3. After mixing PF-127 with sodium hyaluronate evenly, a hydrogel substrate is obtained. S4. Mix the astragaloside A-rADSCs obtained in S2 with the hydrogel substrate obtained in S3, stir, and let stand to remove bubbles to obtain the astragaloside A-rADSCs hydrogel.

[0005] Preferably, the co-culture conditions in S2 are: co-culture at 37°C for 48 hours; the mass concentration of the astragaloside A solution is... .

[0006] Preferably, the final concentration of PF-127 in the astragaloside A-rADSCs hydrogel in S4 is 20 w / v, and the final concentration of sodium hyaluronate is 0.3 w / v.

[0007] Preferably, the stirring conditions in S4 are: stirring at a temperature of 4°C for 15 minutes; and the settling and defoaming time is 20 minutes.

[0008] The present invention also provides the application of the astragaloside A-rADSCs hydrogel prepared by the above preparation method, wherein the astragaloside A-rADSCs hydrogel is used to prepare a drug for repairing diabetic skin damage.

[0009] Compared with the prior art, the present invention has the following advantages: This invention centers on "rabbit adipose mesenchymal stem cells (rADSCs) pretreated with astragaloside A + targeted delivery via thermosensitive hydrogel". It utilizes the active ingredient of astragalus (astragaloside A) to regulate stem cell function and enhance its repair capabilities, avoids serum-related risks through serum-free culture, and achieves controlled release of cytokines using thermosensitive hydrogel, providing a novel, non-invasive, drug-resistant, and highly effective treatment for diabetic skin lesions.

[0010] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0011] Figure 1 This is a graph showing the OD values ​​of P3 generation rADSCs proliferation activity under different mass concentrations of astragaloside A pretreatment for different durations in Example 1 of the present invention.

[0012] Figure 2 This is a graph showing the secretion levels of rADSCs cytokines in the experimental and control groups in Example 1 of this invention.

[0013] Figure 3 This is a diagram showing the repair of diabetic skin lesions in mice using the model group, rADSCs group, and astragaloside A-rADSCs group in Example 2 of the present invention. Detailed Implementation

[0014] Example 1 This embodiment describes a method for preparing astragaloside A-rADSCs hydrogel. The method is as follows: S1. Healthy New Zealand rabbits were selected. After anesthesia, inguinal adipose tissue was dissected, washed, minced, and digested with collagenase for 30 minutes. The tissue was then filtered and centrifuged at 1300 rpm for 5 minutes. Rabbit adipose-derived mesenchymal stem cells (rADSCs) were resuspended in serum-free medium (Yokang Biotechnology, NC0103) and cultured. After primary culture at 37℃ for 48 hours, the medium was changed for the first time. When the cell confluence reached 85%, the cells were passaged at a ratio of 1:3 and cultured to the P3 generation to obtain P3 generation rADSCs. The digestion time and seeding density were strictly controlled throughout the process to ensure high cell purity and stable activity, laying the foundation for subsequent experiments. S2, Combine the P3 generation rADSCs obtained in S1 with a mass concentration of The astragaloside A solution was co-cultured at 37℃ for 48 h, and the supernatant was collected to obtain astragaloside A-rADSCs; S3. After mixing PF-127 (poloxam 407) with sodium hyaluronate evenly, a hydrogel substrate is obtained; S4. The astragaloside A-rADSCs obtained in S2 are mixed with the hydrogel substrate obtained in S3. The mixture is stirred at 4°C for 15 min and allowed to stand for 20 min to remove bubbles, resulting in astragaloside A-rADSCs hydrogel. The hydrogel is liquid at room temperature and easy to apply. It gels within 3 min at 37°C, achieving sustained release of the factor. The final concentration of PF-127 in the astragaloside A-rADSCs hydrogel was 20 w / v, and the final concentration of sodium hyaluronate was 0.3 w / v.

[0015] This embodiment also provides the screening process for the optimal mass concentration of astragaloside A solution and co-culture time in the above preparation method.

[0016] Astragaloside A solution was prepared using serum-free culture medium (Youkang Biotechnology, NC0103) as the solvent. 10 mg of astragaloside A powder was weighed and added to 10 mL of the medium. The solution was then dissolved by vortexing at 2000 rpm for 10 min. Mother liquor, used Sterilization was achieved through membrane filtration, followed by gradient dilution to obtain mass concentrations of [missing values]. (Take 0.2 mL of the stock solution and add 9.8 mL of serum-free culture medium.) (Take 0.4 mL of the stock solution and add 9.6 mL of serum-free culture medium.) (Take 0.6 mL of the stock solution and add 9.4 mL of serum-free culture medium.) (Take 0.8 mL of the stock solution and add 9.2 mL of serum-free culture medium.) (Take 1.0 mL of stock solution and add 9.0 mL of serum-free culture medium) to the target concentration solution; adjust the concentration of P3 generation rADSCs to 1×10⁻⁶. 5 Cells / mL, seeded at 2 mL per well in 6-well plates and pre-cultured for 24 h, then divided into experimental groups ( , , , , The cells were divided into two groups (with the corresponding mass concentration of astragaloside A solution added to each well to ensure a total culture volume of 2 mL per well) and a control group (with an equal volume of serum-free culture medium added). Each group had three replicate wells. All groups were incubated in a 37℃, 5% CO2 incubator. Cell OD values ​​were measured at 24h, 48h, and 72h using the CCK-8 (Cell Counting Kit-8) method.

[0017] like Figure 1 As shown, the co-culture time was 48 hours and the mass concentration was [missing information]. Under the conditions of astragaloside A solution, the OD value of P3 generation rADSCs was significantly higher than that of other groups and the control group (*P<0.05, ***P<0.001), confirming that the co-culture time was 48 h and the mass concentration was [missing value]. The optimal pretreatment conditions were obtained from the astragaloside A solution.

[0018] The astragaloside A-rADSCs obtained in step S2 of this embodiment were used as the experimental group (denoted as positive) and the rADSCs without astragaloside A solution pretreatment were used as the control group (denoted as control). After culturing in serum-free culture medium for 48 h, the supernatant was collected, centrifuged, filtered, and the concentrations of vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) (repair factor), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) (pro-inflammatory factor) were detected by qPCR.

[0019] like Figure 2 As shown, the results indicated that the concentrations of VEGF and IGF in the experimental group were significantly higher than those in the control group (*P<0.05, ***P<0.001), which could effectively promote angiogenesis and collagen synthesis in the wound. Furthermore, the concentrations of TNF-α and IL-6 in the experimental group were significantly lower than those in the control group (***P<0.001), which could effectively reduce the inflammatory response of the wound and improve the healing microenvironment.

[0020] Example 2 This embodiment describes the use of astragaloside A-rADSCs hydrogel prepared in Example 1 for the preparation of a drug for repairing diabetic skin damage.

[0021] The astragaloside A-rADSCs hydrogel obtained in Example 1 was used as the astragaloside A-rADSCs group. The preparation method of the rADSCs hydrogel in this example is the same as that in Example 1, except that the rADSCs of generation P3 in step S2 were not co-cultured with the astragaloside A solution, and the final rADSCs hydrogel was obtained.

[0022] Establishment of a diabetic mouse model and grouping for drug administration: SPF-grade mice aged 6–8 weeks were selected and a diabetic model was established by intraperitoneal injection of streptozotocin (STZ). Mice with blood glucose ≥16.7 mmol / L and stable for 1 week were selected. An 8 mm diameter full-thickness skin defect was created on the back and the mice were randomly divided into 3 groups (18 mice in each group): Model group: applied with physiological saline; rADSCs group: applied with rADSCs hydrogel; Astragalus-rADSCs group: applied with astragaloside A-rADSCs hydrogel prepared in Example 1. All groups were administered 1 mL twice a day, covering the wound with a thickness of about 1 mm. The mice were fed in a routine manner and the experimental conditions were kept consistent.

[0023] like Figure 3 As shown, the wound area in the Astragalus-rADSCs group was significantly smaller than that in the other two groups on day 3; the healing rate was significantly higher in the rADSCs group and the model group on day 7; and the wound was basically closed on day 14, fully demonstrating the effectiveness of the treatment.

[0024] In summary, the astragaloside A-rADSCs hydrogel prepared in Example 1 of this invention not only utilizes the active ingredient of astragalus (astragaloside A) to regulate stem cell function and enhance its repair-promoting ability, but also avoids serum-related risks through serum-free culture, and achieves controlled release of cytokines through thermosensitive hydrogel, and can be used to prepare drugs for the repair of diabetic skin damage.

[0025] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing astragaloside A-rADSCs hydrogel, characterized in that, The method is as follows: S1. Rabbit adipose mesenchymal stem cells were extracted and cultured to generation P3 to obtain generation P3 rADSCs; S2. The P3 generation rADSCs obtained in S1 were co-cultured with astragaloside A solution, and the supernatant was collected to obtain astragaloside A-rADSCs. S3. After mixing PF-127 with sodium hyaluronate evenly, a hydrogel substrate is obtained. S4. Mix the astragaloside A-rADSCs obtained in S2 with the hydrogel substrate obtained in S3, stir, and let stand to remove bubbles to obtain the astragaloside A-rADSCs hydrogel.

2. The method for preparing an astragaloside A-rADSCs hydrogel according to claim 1, characterized in that, The co-culture conditions described in S2 are: co-culture at 37℃ for 48 hours; the mass concentration of the astragaloside A solution is... .

3. The method for preparing an astragaloside A-rADSCs hydrogel according to claim 1, characterized in that, The final concentration of PF-127 in the astragaloside A-rADSCs hydrogel described in S4 is 20 w / v, and the final concentration of sodium hyaluronate is 0.3 w / v.

4. The method for preparing an astragaloside A-rADSCs hydrogel according to claim 1, characterized in that, The stirring conditions in S4 are: stirring for 15 minutes at a temperature of 4°C; and the settling and defoaming time is 20 minutes.

5. The application of an astragaloside A-rADSCs hydrogel prepared by the preparation method according to any one of claims 1-4, characterized in that, The astragaloside A-rADSCs hydrogel is used to prepare a drug for repairing diabetic skin damage.

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